Our goal is to investigate the impact of viral infection on alloreactivity on a human immune system. We will use humanized mice that develop a functional human immune system that can be infected with Epstein Barr Virus (EBV), lymphocytic choriomeningitis (LCMV), or influenza A (lAV), viruses associated with allograft rejection. In C57BL/6 mice, we have shown that heterologous immunity induced by LCMV infection leads to virus-specific allo-cross-reactive CDS T cells that can reject skin allografts and prevent tolerance induced by costimulation blockade. \Ne propose to test the hypothesis in humanized mice that virus infection leads to the generation of human virus specific allo-cross-reactive T cells that participate in rejection of human allografts. Humanized mice will allow analysis of these viruses using novel technologies to quantify naive and effector human alloreactive T cells and virus-induced allo-cross-reactive T cells. These analyses will permit direct determination of their ability to participate in allograft rejection. We also propose use of innovative siRNA in vivo delivery systems to block CD40-CD154 interaction. Aim 1 is to investigate the immune response to alloantigens in human immune system-engrafted NOD-sc/d lL2rf[null] HLA-A2 mice transplanted with human islet or skin allografts. We will quantify the alloimmune response, determine kinetics of graft infiltration and allograft rejection, and establish models for studies of virus-induced heterologous immunity on allograft survival. Aim 2 is to quantify virus-specific and alloimmune responses during acute virus infection in humanized mice treated with costimulation blockade. We will determine human immune responses to EBV, LCMV, and lAV, and quantify virus specific and allospecific T cells that develop as a consequence of heterologous immunity. We will test the hypothesis that virus infection generates heterologous immunity by inducing the development of virus-specific allo-cross-reactive T cells that participate in allograft rejection. These unique resources, novel technologies, and combined expertise in transplantation, humanized mice, virology, and siRNA technology provide an opportunity to investigate the role of heterologous immunity induced by virus infection on alloreactivity and graft rejection in a human immune system.